Caspase Substrate Assay Kit (Colorimetric)

Transcription

1 Caspase Substrate Assay Kit (Colorimetric) Catalog Number KA x 25 assays Version: 02 Intended for research use only

2 Table of Contents Introduction... 3 Background... 3 General Information... 4 Materials Supplied... 4 Storage Instruction... 4 Precautions for Use... 4 Assay Protocol... 5 Assay Procedure... 5 Resources... 6 Troubleshooting... 6 KA / 8

3 Introduction Background Ready-to-use colorimetric substrates for members of caspase family proteases. All substrates were provided in liquid ready-to-use form. KA / 8

4 General Information Materials Supplied List of component Component Caspase-1 Substrate, Ac-YVAD-pNA (4 m/m) Caspase-2 Substrate, Ac-VDVAD-pNA (4 m/m) Caspase-3 Substrate, Ac-DEVD-pNA (4 m/m) Caspase-5 Substrate, Ac-WEHD-pNA (4 m/m) Caspase-6 Substrate, Ac-VEID-pNA (4 m/m) Caspase-8 Substrate, Ac-IETD-pNA (4 m/m) Caspase-9 Substrate, Ac-LEHD-pNA (4 m/m) Amount Storage Instruction Store at -20 C. Shelf life: 1 year under proper storage conditions. Precautions for Use FOR RESEARCH USE ONLY! Not to be used on humans. KA / 8

5 Assay Protocol Assay Procedure 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. Count cells and pellet 1-5 x 10 6 cells. 3. Resuspend cells in 50 µl of chilled cell lysis buffer and incubate cells on ice for 10 minutes. 4. Centrifuge for 1 min in a microcentrifuge (10,000 x g). 5. Transfer supernatant to a fresh tube and assay protein Concentration. 6. Dilute µg protein to 50 µl Cell Lysis Buffer for each assay. 7. Add 50 µl of 2X reaction buffer containing 10 mm DTT to each sample. 8. Add 5 µl of the 4 mm pna conjugated substrates (200 µm final conc.) into each tube individually and incubate at 37 C for 1-2 hour. 9. Read samples at 400- or 405-nm in a microtiter plate reader, or spectrophotometer using a 100-µl micro quartz cuvette (Sigma), or dilute sample to 1 ml with dilution buffer and using regular cuvette (note: Dilution of the samples proportionally decreases the reading). Fold-increase in caspase activity can be determined by comparing these results with the level of the uninduced control. Note: Background reading from cell lysates and buffers should be subtracted from the readings of both induced and the uninduced samples before calculating fold increase in caspase activity. KA / 8

6 Resources Troubleshooting GENERAL TROUBLESHOOTING GUIDE FOR CASPASE COLORIMETRIC AND FLUOROMETRIC KITS: Problems Cause Solution Assay not working Cells did not lyse completely Experiment was not performed at optimal time after apoptosis induction Plate read at incorrect wavelength Old DTT used Resuspend the cell pellet in the lysis buffer and incubate as described in the datasheet Perform a time-course induction experiment for apoptosis Check the wavelength listed in the datasheet and the filter settings of the instrument Always use freshly thawed DTT in the cell lysis buffer High Background Increased amount of cell lysate used Increased amounts of components added due to incorrect pipetting Incubation of cell samples for extended periods Use of expired kit or improperly stored reagents Contaminated cells Refer to datasheet and use the suggested cell number to prepare lysates Use calibrated pipettes Refer to datasheet and incubate for exact times Always check the expiry date and store the individual components appropriately Check for bacteria/ yeast/ mycoplasma contamination KA / 8

7 Lower signal Cells did not initiate apoptosis Determine the time-point for initiation of levels Very few cells used for analysis apoptosis after induction (time-course Use of samples stored for a long experiment) time Refer to datasheet for appropriate cell Incorrect setting of the equipment number used to read samples Use fresh samples or aliquot and store Allowing the reagents to sit for and use within one month for the assay extended times on ice Refer to datasheet and use the recommended filter setting Always thaw and prepare fresh reaction mix before use Samples with erratic readings Uneven number of cells seeded in the wells Samples prepared in a different buffer Adherent cells dislodged and lost at the time of experiment Cell/ tissue samples were not completely homogenized Samples used after multiple freeze-thaw cycles Presence of interfering substance in the sample Use of old or inappropriately stored samples Seed only equal number of healthy cells (correct passage number) Use the cell lysis buffer provided in the kit Perform experiment gently and in duplicates/triplicates; apoptotic cells may become floaters Use Dounce homogenizer (increase the number of strokes); observe efficiency of lysis under microscope Aliquot and freeze samples, if needed to use multiple times Troubleshoot as needed Use fresh samples or store at correct temperatures until use Unanticipated Measured at incorrect wavelength Check the equipment and the filter results Cell samples contain interfering setting substances Troubleshoot if it interferes with the kit (run proper controls) KA / 8

8 General issues Improperly thawed components Incorrect incubation times or temperatures Incorrect volumes used Air bubbles formed in the well/tube Substituting reagents from older kits/ lots Use of a different 96-well plate Thaw all components completely and mix gently before use Refer to datasheet & verify the correct incubation times and temperatures Use calibrated pipettes and aliquot correctly Pipette gently against the wall of the well/tubes Use fresh components from the same kit Fluorescence: Black plates; Absorbance: Clear plates Note: The most probable cause is listed under each section. Causes may overlap with other sections. KA / 8